Project Summary/Abstract: The efficacy of injected therapeutic proteins is often compromised by their rapid clearance from the circulation. This means that doses must be administered more often, impacting both patient compliance and cost of treatment. A major search is underway in academic and industrial laboratories to discover ways to modify protein drugs in such a way that their residence time in the body is enhanced. Approaches include chemical modification of the protein therapeutic, or creation of protein drugs that contain stretches of additional amino acids, or indeed, of whole proteins, designed to promote increased half-life. The long-term objective of the present proposal is to provide a means that is applicable in principle to all therapeutic proteins for extending their residence time in the body, thus making them easier to administer and lower in cost to the patient. In this application we propose to explore the introduction of peptide sequences, sequence tags that will allow for limited glycosylation of the protein therapeutic during its recombinant production in a mammalian cell line. The hypothesis is that this vehicle for enhancing glycosylation will protect the protein drug from rapid elimination. Our novel sequence tags are derived from regions in a human protein that are known to undergo glycosylation and that should allow for incorporation of carbohydrate to enhance the lifetime of the protein drug in the body. The specific aims of the proposal are 1. To clone and express in HEK-293 cells, N- and C-terminally extended variants of a well-studied therapeutic protein in which the extensions comprise human protein sequences, varying in length and known to undergo glycosylation;2. To purify the proteins to homogeneity and verify that they contain levels of carbohydrate that correspond to the length of the adduct;3. To show that the N- and/or C-terminal extensions have not abolished biological activity;and 4. To test these tagged proteins in animal models to see if they increase serum half-life relative to the native parent protein drug. The research plan will be designed to address these specific aims and will combine expertise with methods of molecular and cell biology for production and expression of the new gene constructs in HEK-293 cells, together with expertise in protein purification and characterization to produce viable proteins for testing. PUBLIC HEALTH RELEVANCE: Project Narrative :The complexities of many unsolved and unmet medical needs such as cancer, Alzheimer's disease, ALS, and other neurodegenerative disorders has increased focus on development of proteins as drugs to treat these maladies. Protein drugs are expensive due to high cost of manufacture, and they tend to be rapidly eliminated from the body, thus increasing the dose regimen and adding to the price of treatment. The present proposal provides a novel method, applicable in principle to any protein drug, for producing variants with increased duration of activity (fewer doses), and reduced cost.